Methods and compositions involving whey protein isolates

ABSTRACT

The present invention concerns methods of isolating milk proteins. Methods of the invention include charged ultrafiltration processes that use variations in pH to further separate protein species.

This is a continuation of co-pending application Ser. No. 13/181,234,filed Jul. 12, 2011, which claims benefit of priority to provisionalapplication Ser. No. 61/365,653, filed Jul. 19, 2010, the entirecontents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates generally to the field of proteinchemistry. More particularly, it provides a process of separating milkproteins using charged ultrafiltration.

Description of Related Art

Purified dairy proteins have special, value-added utility in processedfoods and medical foods. For example, glycomacropeptide (GMP) is aprotein present in cheese whey that is the only protein in nature thatis safe to eat for individuals with phenylketonuria. Alpha-lactalbumin(ALA) is a protein present in milk and whey that has utility in infantformula; mother's milk has 130% more ALA than cow's milk.

Furthermore, ALA has four times more tryptophan (Trp) than an averagewhey protein. Trp is a precursor to the neurotransmitter serotonin inthe brain that controls appetite, depression, mood, and sleep.

The value of purified dairy proteins is much greater than a mixture ofthe dairy proteins. For example, whey protein isolate is a mixture ofwhey proteins and sells for about $10/kg. GMP sells for about $70/kg andALA for about $50/kg. Thus, there is a human need and economic benefitto the fractionation of dairy proteins.

Chromatography has been used traditionally to fractionate dairyproteins. Ion exchange chromatography is used to manufacture GMP and ALAfrom whey. Chromatography is expensive and not environmentally friendlybecause of the need for sophisticated chromatography systems and thedisposal of waste water and buffers. The dairy industry has been slow toadopt chromatography for these reasons and because it is unfamiliar toproduction personnel. The inventor has examined the use ofpositively-charged membranes to increase the selectivity ofultrafiltration and allow the fractionation of proteins from cheesewhey. By adding a positive charge to ultrafiltration membranes, andadjusting the solution pH, it was possible to permeate proteins havinglittle or no charge, such as glycomacropeptide, and retain proteinshaving a positive charge. Placing a charge on the membrane increased theselectivity by over 600% compared to using an uncharged membrane. Thedata were fit using the stagnant film model that relates the observedsieving coefficient to membrane parameters such as the flux, masstransfer coefficient, and membrane Peclet number. However, this approachwas not tested for other species, such as those in milk, nor in thecontext of large-scale separation. Thus, improved industrial methods forseparating milk proteins are needed.

SUMMARY OF THE INVENTION

Thus, in accordance with the present invention, there is provided amethod for fractionating a protein mixture comprising multiple proteinspecies to obtain a protein of interest comprising (a) adjusting the pHof said protein mixture based on the isoelectric point of said proteinof interest, thereby rendering a net charge of about zero on saidprotein of interest, (b) adjusting the conductivity of said proteinmixture such that shielding of said multiple species other than saidprotein of interest is limited to the extent that said multiple speciesother than said protein of interest are rejected by a chargedultrafiltration membrane; (c) contacting said mixture with said chargedultrafiltration membrane to achieve a first permeate and a firstretentate, wherein said ultrafiltration membrane has a pore size atleast 100 kDa above, or 10× greater than, at least one of said multiplespecies other than said protein of interest, wherein said first permeatecomprises an increased ratio of said protein of interest as compared tosaid protein mixture. The protein mixture may be a milk protein or awhey protein mixture. The charged ultrafiltration may be effected by anultrafiltration membrane having a pore size rating of 150-500 kDa, suchas 150 kDa, 175 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450kDa or 500 kDa. The protein mixture may comprise one or more ofglycomacropeptide (GMP), alpha-lactalbumin (ALA), immunoglobulin G(IgG), and/or beta-lactoglobulin (BLG).

The method may further comprise subjecting said first permeate to asecond charged ultrafiltration to achieve a second permeate and a secondretentate, and the second retentate may be recycled into another proteinmixture for additional charged ultrafiltration. The method may alsofurther comprise subjecting said first retentate to a second chargedultrafiltration to achieve a second retentate and a second permeate, andwherein said second permeate may be recycled into another proteinmixture for additional charged ultrafiltration. The ultrafiltration mayachieve a purity of about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and99%, and may achieve a purity of about 60%, 65%, 70%, 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99%. The method may be effected by a multistagecrossed-flow positively-charged of ultrafiltration membrane.

The ultrafiltration membrane may be positively-charged ornegatively-charged. The method may further comprise adjusting the pH ofsaid protein mixture to minimize the charge on said protein of interest,such as pH 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5,4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9,6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3,7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8, 8.9, or 9.0, and in particular, pH 3-6, 3-5, 3-4, 4-6, 5-6, or 4-5.The method may further comprise adjusting the conductivity of saidprotein mixture to 3-10 mS/cm, including 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9,5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3,6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1,9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0 mS/cm, and in particular,3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 5-10, 6-10, 7-10, 8-10, or9-10. The proteins separated may be GMP from ALA, GMP from IgG, GMP fromBLG, ALA from IgG, ALA from BLG, or BLG from IgG. In some embodimentsthe methods of the invention involve implementing separation of proteinsin a batch process. The term “batch” is used according to its ordinaryand plain meaning in this field to refer to a process in whichcomponents of the purification process are incubated together, generallywithout regard to order or direction.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein. Moreover, it is clearly contemplated that embodiments may becombined with one another, to the extent they are compatible.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

It is specifically contemplated that any embodiments described in theExamples section are included as an embodiment of the invention.

Following the long-standing patent law convention, the words “a” and“an,” when used in conjunction with the word “comprising” in the claimsor specification, denotes one or more, unless specifically noted.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Effect of charge, pH and conductivity.

FIG. 2. Flow diagram for two-stage charged ultrafiltration membrane.

FIG. 3. Two-stage charged ultrafiltration of ALA and BLG.

FIG. 4. Flow diagram for three-staged charged ultrafiltration membrane.

FIG. 5. Milk serum protein fractionation using a positively-charged 300kDa ultrafiltration membrane.

FIG. 6. Two-stage charged ultrafiltration of MSP.

FIG. 7. Two-stage charged ultrafiltration of MSP.

FIG. 8. Stages with and without recycle.

FIG. 9. Glycomacropeptide (GMP) separation from Swiss cheese whey.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present inventors have discovered that charged ultrafiltrationmembranes can be used to make dairy protein fractions of chromatographicpurity without the need for sophisticated chromatography equipment orwater or buffers. The invention can be practiced using ultrafiltrationmembrane systems common in essentially every dairy processing facilityworldwide. The inventors made the surprising discovery that chargedultrafiltration membranes having a pore size rating of 150-500 kDa canbe used to fractionate dairy proteins much smaller in size such as GMP(8.6 kDa), ALA (14.4 kDa), and beta-lactoglobulin (18.6 kDa). They alsofound that high purity and yield can be 10 attained by operation of themembranes in stages analogous to plates in a distillation column.

I. PROTEINACEOUS COMPOSITIONS

In certain embodiments, the present invention concerns proteincompositions comprising at least one proteinaceous molecule, such as awhey protein. As used herein, a “proteinaceous molecule,” “proteinaceouscomposition,” “proteinaceous compound,” “proteinaceous chain” or“proteinaceous material” generally refers, but is not limited to, aprotein of greater than about 50 amino acids or the full lengthendogenous sequence translated from a gene; a polypeptide of greaterthan about 100 amino acids; and/or a peptide of from about 3 to about100 amino acids. All the “proteinaceous” terms described above may beused interchangeably herein.

A. Milk Proteins:

There are several types of proteins in milk. The major milk proteins areunique to milk. They are not found in any other tissue. Milk proteins,particularly caseins, have an appropriate amino acid composition forgrowth and development of the young. Other proteins in milk include anarray of enzymes, proteins involved in transporting nutrients, proteinsinvolved in disease resistance (antibodies and others), growth factors,etc.

The total protein component of milk is composed of numerous specificproteins. The primary group of milk proteins are the caseins. There are3 or 4 caseins in the milk of most species; the different caseins aredistinct molecules but are similar in structure. All other proteinsfound in milk are grouped together under the name of whey proteins. Themajor whey proteins in cow milk are beta-lactoglobulin andalpha-lactalbumin.

The major milk proteins, including the caseins, beta-lactoglobulin andalpha-lactalbumin, are synthesized in the mammary epithelial cells andare only produced by the mammary gland. The immunoglobulin and serumalbumin in milk are not synthesized by the epithelial cells. Instead,they are absorbed from the blood (both serum albumin and theimmunoglobulins). An exception to this is that a limited amount ofimmunoglobulin is synthesized by lymphocytes which reside in the mammarytissue (called plasma cells). These latter cells provide the mammarygland with local immunity. Milk proteins can be identified by molecularmass. The relative size of the caseins (˜25-35 kDa) is distinguishedfrom the major whey proteins beta-lactoglobulin (18 kDa) andalpha-lactalbumin (14 kDa). Others include primarily lactoferrin (˜80kDa) and serum albumin (˜66 kDa).

B. Caseins:

Caseins have an appropriate amino acid composition that is important forgrowth and development of the nursing young. This high quality proteinin cow milk is one of the key reasons why milk is such an importanthuman food. Caseins are highly digestible in the intestine and are ahigh quality source of amino acids. Most whey proteins are relativelyless digestible in the intestine, although all of them are digested tosome degree. When substantial whey protein is not digested fully in theintestine, some of the intact protein may stimulate a localizedintestinal or a systemic immune response. This is sometimes referred toas milk protein allergy and is most often thought to be caused bylactoglobulin. Milk protein allergy is only one type of food proteinallergy.

Caseins are composed of several similar proteins which form amulti-molecular, granular structure called a casein micelle. In additionto casein molecules, the casein micelle contains water and salts (mainlycalcium and phosphorous). Some enzymes are associated with caseinmicelles as well. The micellar structure of casein in milk is animportant part of the mode of digestion of milk in the stomach andintestine, the basis for many of the milk products industries (such asthe cheese industry), and the basis for the ability to easily separatesome proteins and other components from cow milk. Casein is one of themost abundant organic components of milk, in addition to the lactose andmilk fat. Individual molecules of casein alone are not very soluble inthe aqueous environment of milk. However, the casein micelle granulesare maintained as a colloidal suspension in milk. If the micellarstructure is disturbed, the micelles may come apart and the casein maycome out of solution, forming the gelatinous material of the curd. Thisis part of the basis for formation of all non-fluid milk products likecheese.

Caseins are highly digestible in the intestine and are a high-qualitysource of amino acids. Most whey proteins are relatively less digestiblein the intestine, although all of them are digested to some degree. Whensubstantial whey protein is not digested fully in the intestine, some ofthe intact protein may stimulate a localized intestinal or a systemicimmune response. This is sometimes referred to as milk protein allergyand is most often thought to be caused by B-lactoglobulin. Milk proteinallergy is only one type of food protein allergy.

C. Whey Proteins:

Whey proteins comprise one of the two major protein groups of bovinemilk and account for approximately 20% of the milk composition. However,the present invention is not limited to whey protein from bovine milkand can be implemented with respect to the milk from other species. Wheyprotein is derived as a natural byproduct of the cheese-making process.In addition to proteins, the raw form contains fat, lactose and othersubstances. The raw form is processed to produce protein-rich wheyprotein concentrates (WPC) and whey protein isolates (WPI), among otherthings. Thus, whey proteins are comprised of high-biological-valueproteins and proteins that have different functions. The primary wheyproteins are beta-lactoglobulin and alpha-lactalbumin, two smallglobular proteins that account for about 70 to 80% of total wheyprotein. Proteins present in lesser amounts include the immunoglobulinsIgG, IgA and IgM, but especially IgG, glycomacropeptides, bovine serumalbumin, lactoferrin, lactoperoxidase and lysozyme.

There are many whey proteins in milk and the specific set of wheyproteins found in mammary secretions varies with the species, the stageof lactation, the presence of an intramammary infection, and otherfactors. The major whey proteins in cow milk are beta-lactoglobulin andalpha-lactalbumin. Alpha-lactalbumin is an important protein in thesynthesis of lactose and its presence is central to the process of milksynthesis. beta-lactoglobulin's function is not known. Other wheyproteins are the immunoglobulins antibodies; especially high incolostrum) and serum albumin (a serum protein). Whey proteins alsoinclude a long list of enzymes, hormones, growth factors, nutrienttransporters, disease resistance factors, and others.

D. Milk Serum Proteins:

Microfiltration of milk removes the casein micelles in the retentate andleaves the non-casein proteins of milk in the permeate. When the caseinsare removed from milk without making cheese, the remaining proteins arecomprised of the proteins found in whey with the exception ofglycomacropeptide. The action of rennet or chymosin on kappa-caseincleaves off the hydrophilic glycomacropeptide, leaving the hydrophobicpara-kapa-casein to coagulate and form cheese curd. When this enzymaticcleavage does not occur, glycomacropeptide generation also does notoccur. Thus, the proteins in the milk microfiltration permeate arecalled milk serum proteins instead of whey proteins to highlight thedistinction in composition, namely the absence of glycomacropeptide inmilk serum proteins.

II. ULTRAFILTRATION

Ultrafiltration (UF) is a variety of membrane filtration in whichhydrostatic pressure forces a liquid against a semipermeable membrane.Suspended solids and solutes of high molecular weight are retained,while water and low molecular weight solutes pass through the membrane.This separation process is used in industry and research for purifyingand concentrating macromolecular (103-106 Da) solutions, especiallyprotein solutions. Ultrafiltration is not fundamentally different frommicrofiltration or nanofiltration, except in terms of the size of themolecules it retains. Ultrafiltration is applied in crossflow ordead-end mode and separation in ultrafiltration undergoes concentrationpolarization.

Ultrafiltration systems eliminate the need for clarifiers and multimediafilters for waste streams to meet critical discharge criteria or to befurther processed by wastewater recovery systems for water recovery.Efficient ultrafiltration systems utilize membranes which can besubmerged, back-flushable, air scoured, spiral wound UF/MF membrane95091326. that offers superior performance for the clarification ofwastewater and process water. There are a number of different formats ofultrafiltration membrane geometries:

-   -   Spiral wound module: consists of large consecutive layers of        membrane and support material rolled up around a tube; maximizes        surface area; less expensive, however, more sensitive to flux        decline caused by accumulation of solutes on the membrane.    -   Tubular membrane: Feed solution flows through the membrane lumen        and the permeate is collected in the tubular housing; generally        used for viscous or crude fluids; system is not very compact and        has a high cost per m2 installed.    -   Hollow fiber membrane: Modules contain several small (0.6 to 2        mm diameter) tubes or fibers; feed solution flows through the        lumens of the fibers and the permeate is collected in the        cartridge area surrounding the fibers; filtration can be carried        out either “inside-out” or “outside-in.”        Module configurations include:    -   Pressurized system or pressure-vessel configuration: TMP        (transmembrane pressure) is generated in the feed stream by a        pump, while the permeate stays at lower pressure closer to        atmospheric pressure. Pressure-vessels are generally        standardized, allowing the design of membrane systems to proceed        independently of the characteristics of specific membrane        elements.    -   Immersed system: Membranes are suspended in basins containing        the feed and open to the atmosphere. Pressure on the influent        side is limited to the pressure provided by the feed column. TMP        is generated by a pump that develops suction on the permeate        side. Ultrafiltration, like other filtration methods can be run        as a continuous or batch process.

A negatively charged membrane can be obtained by sulfonation ofpolysulfone, and a positively charged polymer can be synthesized bychloromethylation of polysulfone and then by quaternization of the aminogroup. U.S. Patent Publication 2003/0178368 A1 teaches how to make acharged cellulosic filtration membrane by covalently modifying themembrane's surfaces with a charged compound or a compound capable ofbeing chemically modified to possess a charge. For example, a cellulosic(cellulose, cellulose di- or tri-acetate, cellulose nitrate or blendsthereof) membrane has hydroxyl moieties that are derivatized to form thecharged surfaces. A wide variety of compounds can be used. Most possessa halide moiety capable of reacting with the membrane surface (includingthe interior of its pores) as well as a hydroxyl moiety capable ofreacting with a second ligand that imparts the charge, positive ornegative.

U.S. Pat. No. 4,824,568 teaches casting a polymeric coating onto amembrane's surface and then cross-linking it in place with UV light,electron beam or another energy source to input a charge to the membranesuch as PVDF, polyethersulfone, polysulfone, PTFE resin and the like.Examples of charged membranes are also found in U.S. Pat. No. 4,849,106and U.S. Patent Publication 2002/0185440.

III. ADJUSTING PH TO EFFECT PERMEATION

Adjusting the pH of the protein mixture feed stream to the chargedultrafiltration membrane is key to fractionation of proteins.Fractionation requires that one protein permeate the membrane more thananother protein. Charged ultrafiltration is different than traditionalultrafiltration in that the charge of the protein relative to the chargeof the membrane is a key factor in addition to the size of the proteinrelative to the pore size of the membrane. Generally, when the pH of thesolution is less than the isoelectric point (pl) of a protein, then theprotein has a net positive charge. Conversely, when the pH is above theisoelectric point, then the protein has a net negative charge. In orderfor a charged ultrafiltration membrane to permeate a protein of interestand not the other proteins in the mixture it is desired to have theprotein of interest be relatively smaller and uncharged compared to theother proteins.

For example, milk serum proteins can be made by microfiltration of milkto remove the caseins. The milk serum protein contains predominately theproteins alpha-lactalbumin and beta-lactoglobulin. Alpha-lactalbumin issmaller (14.4 kDa) than beta-lactoglobulin (18.6 kDa) and is more acidic(pl 4.4) than beta-lactoglobulin (pl 5.1). By adjusting the milk serumprotein to about pH 4.0 to 4.5, the alpha-lactalbumin has little to nonet charge while the beta-lactoglobulin has a net charge that ispositive. The larger beta-lactoglobulin will be subject to electrostaticrepulsion by a positively-charged ultrafiltration membrane while thesmaller alpha-lactalbumin that has little to no net charge can permeatethe charged ultrafiltration membrane.

In another example, cheese whey contains predominatelyglycomacropeptide, alpha-lactalbumin, and beta-lactoglobulin.Glycomacropeptide is smaller (8.6 kDa) and more acidic (pl<3.8) than theother whey proteins. By adjusting the whey to pH 3 to 4, theglycomacropeptide has little to no net charge while alpha-lactalbuminand beta-lactoglobulin have a net charge that is positive. At pH 3 to 4,the other proteins in whey that are larger than glycomacropeptide willbe subject to electrostatic repulsion by a positively chargedultrafiltration membrane while glycomacropeptide is not. Thus, adjustingthe whey to pH 3 to 4 effects the permeation of glycomacropeptide.

IV. ADJUSTING CONDUCTIVITY TO MINIMIZE SHIELDING

Increasing the conductivity of the protein mixture increases shieldingof the charges on the proteins. As conductivity increases to above about100 mS/cm, charge shielding is great enough to negate the effect ofelectrostatic repulsion. This is undesirable because it takes away theadvantages of charged ultrafiltration membranes compared to traditionalultrafiltration membranes. Conversely, lowering the conductivity isundesirable for dairy protein fractionation because milk and whey have anatural conductivity of about 3 to 10 mS/cm. Lowering the conductivityby diafiltration or electrodialysis is expensive.

Dissolving the dry dairy proteins in a dilute buffer solution is anothercommonly used method to adjust the pH and operate at low conductivity.This is undesirable however, because buffer salts are expensive and ahazard to the environment. Furthermore, drying the dairy proteins isexpensive, and adding water and buffer to the dry proteins prior tofractionation by charged ultrafiltration is an unnecessary and imprudentextra step. It is desired to fractionate dairy proteins from the milk orwhey or milk serum protein stream without the addition of buffer saltsor the adjustment of the milk or whey to a conductivity substantiallylower than the natural value.

The inventors have found that there is a balance between pH andconductivity. Increasingly lowering the pH to less than the isoelectricpoint of a protein generally increases the positive charge on theprotein. That increase in positive charge counteracts the chargeshielding effect of increasing the conductivity. Therefore, to operateat the high conductivity natural to milk and whey, the inventors havefound that the pH of the solution can be lowered to ameliorate chargeshielding.

Thus, in accordance with the present invention, the inventors proposedadjusting the conductivity to a range of about 3-10 mS/cm and a pH of3-4.5. This will avoid any significant charge shielding, but can beaccomplished in a fairly straightforward manner. If they had chosen tolower the conductivity to, say, less than 2 mS/cm, then charge shieldingwould be minimal. That would enhance the effect of electrostaticrepulsion and one would not need to go reduce substantially pH, i.e.,the difference between pH and pl could have been smaller. The bigger thedifference between pH and pl the bigger the net charge on the protein.Reducing the conductivity to less than 2 mS/cm would have lessened thecost to reduce the pH, but would have increased more the cost forconductivity reduction. It is generally less expensive to reduce the pHthan to reduce the conductivity of the whey or milk serum.

By choosing to work at the natural conductivity of milk and whey, theissue was made more difficult because of enhanced chargeshielding—charge shielding negates the advantages of a chargedultrafiltration. To ameliorate this problem, the inventors adjusted thepH to below the value one might have been able to use were one workingat a lower conductivity. This increased the net charge on the proteinsto be rejected from the membrane while maintaining the naturalconductivity of milk and whey.

One can adjust the conductivity independent of the pH. Adding sodiumchloride or another neutral salt increases the conductivity withoutchanging the pH, and adding deionized water lowers the conductivitywithout changing the pH.

V. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1

A 300 kDa cross-flow ultrafiltration membrane (50 cm² regeneratedcellulose membrane, Pellicon XL, Millipore, Bedford, Mass., USA) wasused to separate alpha-lactalbumin (ALA) and beta-lactoglobulin (BLG) toillustrate the effect of membrane charge, and solution pH andconductivity on the observed sieving coefficient (S₀) and selectivity ofthe separation (FIG. 1). An uncharged 300 kDa ultrafiltration membraneshould not retain ALA (14.4 kDa) and BLG (18.6 kDa) because bothproteins are at least ten times smaller than the rated membrane poresize. As expected, this is what was observed. Most of the ALA and BLGfreely passed through the uncharged membrane, with only about 8 to 16%of the ALA and 15 to 19% of the BLG retained at pH 4.0 to 4.5 andconductivity 4 to 6 mS/cm (S₀ ALA 0.88, S₀ BLG 0.83). Selectivity of theseparation is defined as the ratio of the sieving coefficients and noseparation occurs for a selectivity of one. The selectivity of theuncharged ultrafiltration membrane for ALA and BLG was about 1.0. Thus,ALA and BLG were not significantly retained by the 300 kDa unchargedultrafiltration membrane, and it failed to separate one protein from theother.

A positive charge was covalently bonded to the 300 kDa ultrafiltrationmembrane surface using (3-bromopropyl)trimethylammonium bromidefollowing the procedure of Bushan and Etzel (2009) and van Reis (2006).Placing a positive charge on the 300 kDa membrane dramatically increasedthe selectivity of the separation compared to the uncharged membrane by330% (FIG. 1). Furthermore, increasing the solution conductivityincreased the sieving coefficient for ALA, but not for BLG. Thisincreased the selectivity of the separation by 70% for the chargedmembrane (from 2.5 to 4.3) as conductivity increased from 4 to 8 mS/cm.

Increasing pH from 4.0 to 4.5 increased the sieving coefficients forboth ALA and BLG, and decreased the selectivity. However, anintermediate pH of 4.3 was found to result in the best separation,because it increased the selectivity when compared to pH 4.0 or 4.5.Thus, the best conditions for separation of ALA from BLG were found tobe pH 4.3 and conductivity 8 mS/cm using a positively charged 300 kDaultrafiltration membrane. Under these conditions, the selectivity was5.7 using the charged ultrafiltration membrane; 470% greater than forthe uncharged membrane.

If ALA is the protein of interest, and the other species is BLG, thenadjusting the solution to pH 4.3, near the isoelectric point of ALA (pl4.4), but substantially less than the isoelectric point of BLG (pl 5.1),thereby rendering a net charge of about zero on the ALA and asubstantial positive charge on the BLG, and adjusting the solutionconductivity to about 8 mS/cm, the protein of interest ALA (14.4 kDa)could be fractionated from BLG (18.6 kDa) using a chargedultrafiltration membrane having a pore size rating at least 100 kDaabove, or ten-fold greater than the protein species other than theprotein of interest. This was not possible using an unchargedultrafiltration membrane, because both of the proteins freely passedthrough the uncharged membrane because it had a pore size rating morethan ten times larger than either of the proteins. What is surprisingabout this result is that by simply adding a charge to theultrafiltration membrane, proteins more than ten times smaller than therated pore size of membrane can be fractioned from each other.

Example 2

The conditions of Example 1 can be used on a feed stream (FS) to make afirst permeate stream (P1) enriched in the protein of interest (ALA) anda first retentate stream (R1) enriched in the other species (BLG). Asshown in FIG. 2, a second charged ultrafiltration membrane can be usedon the first retentate stream to make a second permeate stream (P2) anda second retentate stream (R2). This staging of the chargedultrafiltration membranes has the advantage of increasing purity andoffering the opportunity to recycle intermediate streams to the feedstream (FS) to reduce waste.

For example, a feed stream containing 0.5 g/L each of ALA and BLG wasadjusted to pH 4.3, conductivity 8 mS/cm and fed to a 300 kDa chargedultrafiltration membrane (FIG. 3). The feed stream had 50% purity eachfor ALA and BLG. The first permeate stream (P1) was higher in ALA purity(77%) and the first retentate stream (R1) was higher in BLG purity (68%)than the feed stream (FS). The first permeate stream was fed to a second300 kDa charged ultrafiltration membrane. The second permeate (P2) wasfurther enriched in ALA (87% purity) and the second retentate stream(R2) had a composition and purity about the same as the feed stream, andcould have been be recycled back to the feed stream to recover theproteins and reduce waste as shown in FIG. 3.

The first retentate stream (R1) could be subjected to a third chargedultrafiltration to make a third retentate (R3) and a third permeate (P3)as shown in FIG. 4. The third retentate in this case would be enrichedin BLG compared to the first retentate, and the third permeate wouldhave a composition similar to the feed stream, and could be recycledback to the feed stream to recover the proteins and reduce waste.

Example 3

Milk serum protein (MSP) was fractionated using a charged 300 kDaultrafiltration membrane. Skim milk was subjected to microfiltration toremove caseins and residual lipids, and form the MSP solution. The MSPsolution was adjusted to pH 4.3 without adjustment of the conductivity.The natural conductivity of the MSP solution was about 8-10 mS/cm. Itwas desired to fractionate the MSP at this natural conductivity withoutthe use of either electrodialysis, diafiltration, dilution by additionof de-ionized water or other methods to lower the conductivity. Usingthe MSP at the natural conductivity is less expensive than the use ofany of the preceding methods. It was also desired to avoid thedissolution of dried MSP in buffer to eliminate the expensive steps ofconcentration and drying of the MSP, and the expense of addition andthen disposal of the buffer salts. By using the natural MSP solutionthat was made directly from the microfiltration of skim milk, the costof manufacture and the amount of waste generation are less than theseother methods.

As shown in FIG. 5, the sieving coefficients for ALA and BLG using the300 kDa uncharged (UNCHG) ultrafiltration membrane were notstatistically significantly different (p>0.05). The selectivity wasabout 1.4, meaning there was little to no separation of ALA and BLG.Placing a positive charge on the 300 kDa ultrafiltration membrane (CHG)increased the selectivity by 66% to a value of 2.2. To further increasethe selectivity, the temperature was increased from 22 to 40° C. Thisincreased the selectivity of the MSP separation from 2.2 to 4.2.

A two-stage separation was conducted using MSP at 40° C. and pH 4.3(FIG. 6). The MSP feed stream contained 1.2 g/L ALA at 35% purity and2.2 g/L BLG at 65% purity. The first permeate stream (P1) was higher inALA purity (55%) and the first retentate stream (R1) was higher in BLGpurity (77%) compared to the feed stream. The first permeate stream wasfed to a second 300 kDa charged ultrafiltration membrane. The secondpermeate (P2) was further enriched in ALA (82% purity) and the secondretentate stream (R2) had a composition and purity about the same as thefeed stream, and could have been recycled back to the feed stream torecover the proteins and reduce waste. Thus, ALA and BLG werefractionated from MSP without the addition of buffer salts or the needto reduce the solution conductivity.

Example 4

The impacts of multiple stages and recycle on concentrations and puritycan be illustrated using a mass balance calculation. The measuredconcentrations for the two-stage system without recycle were compared tothe mass balance calculations (FIG. 7).

Measured values for the sieving coefficients at 40° C. were used in thecalculation (S₀ ALA=0.520, S₀ BLG=0.125). The mass balance calculationswere not substantially different than the experimental measurements.

Four different flow configurations were evaluated: (1) one stage, (2)two stage, (3) two stage with recycle, and (4) three stage with recycle(FIG. 8). Concentrations of each stream for a feed stream of MSP weredetermined by mass balance. In the two stage system, the first permeate(P1) was fed to a second membrane to form a second permeate (P2) andsecond retentate (R2). For two stages with recycle, stream R2 wasrecycled back to the feed stream. The composition of stream R2 wassimilar to the composition of the feed stream. The three stage systemwas the same as the two stage system in the handling of stream P1, butadded that the first retentate (R1) was fed to a third membrane to forma third permeate (P3) and third retentate (R3). Streams R2 and P3 wererecycled back to the feed stream in the calculation for the three cyclesystem because the compositions were similar and it reduced waste.

Purity of ALA increased by adding more stages and recycle (Table 1).Purity of BLG was less affected, but was highest for three stages withrecycle. The ALA purity of the feed stream was 35% compared to a purityof 88% in the top of the three-stage system with recycle. This exampleshows that positively-charged ultrafiltration membranes can be used tomake dairy protein fractions of chromatographic purity without the needfor sophisticated chromatography equipment or water or buffers. Theinvention can be practiced using ultrafiltration membrane equipmentalready in place in essentially every dairy processing facilityworldwide.

TABLE 1 Purity for Different Flow Configurations ALA BLG purity purityin top in bottom 1 stage 62% 78% 2 stage no recycle 83% 78% 2 stage withrecycle 86% 76% 3 stage with recycle 88% 83%

Example 5

A 300 kDa positively charged ultrafiltration membrane was used toseparate glycomacropeptide (GMP) from alpha-lactalbumin (ALA) andbeta-lactoglobulin (BLG) in Swiss cheese whey at pH 3.0 and conductivity4 mS/cm. It was expected that GMP (8.6 kDa, pl<3.8) would be smaller andless charged than the other major proteins in whey that are larger andmore basic ALA (14.4 kDa, PI=4.4) and BLG (18.6 kDa, pl=5.1). Therefore,GMP might be expected to freely pass through a positively-chargedultrafiltration membrane while ALA and BLG would not. What was notexpected was that it would be possible to use a 300 kDa membrane poresize. It was surprising to observe that both ALA and BLG, which are atleast ten times smaller than the rated membrane pore size, were highlyretained by the 300 kDa charged membrane. The sieving coefficients were:S₀ GMP=0.43, S₀ BLG=0.105, and S₀ ALA=0.133. The positively chargedmembrane retained about 90% of the BLG and 87% of the ALA. The proteinof interest (GMP) more freely passed through the membrane.

For a single stage, the measured concentrations of GMP, BLG, and ALAwere compared to those from the mass balance calculation (FIG. 9).Measured and calculated concentrations were not significantly different.The calculated concentrations for a three stage configuration withrecycle of streams P3 and R2 revealed an increase in purity of the GMPfrom 57% in the feed stream to 80% in the permeate (P1) of the one-stagesystem, and 94% in the permeate (P2) of the three stage system.

This example illustrates that if GMP is the protein of interest, and theother species are the other major whey proteins BLG and ALA, thenadjusting the cheese whey solution to pH 3.0, near the isoelectric pointof GMP, but substantially less than the isoelectric point of BLG andALA, renders a net charge of about zero on the GMP and a substantialpositive charge on the BLG and ALA. Using a charged ultrafiltrationmembrane having a pore size rating at least 100 kDa above, or ten-foldgreater than the protein species other than the protein of interest,means that the protein of interest GMP (8.6 kDa) could be collected inthe permeate and separated from BLG (18.6 kDa) and ALA (14.4 kDa) whichare collected in the retentate.

In the previous examples, ALA was the protein of interest and theprotein mixture was adjusted to pH 4.3 to render a net charge of aboutzero on ALA while the other proteins carried a substantial net positivecharge. This allowed ALA to freely pass through the 300 kDa positivelycharged ultrafiltration membrane while the other proteins did not. Inthe present example, ALA was not the protein of interest (GMP was) andby adjusting the protein mixture to pH 3.0, ALA was substantiallyrejected by the same 300 kDa charged ultrafiltration membrane thatallowed ALA to freely pass through at pH 4.3.

BLG was not the protein of interest in any of the examples. At both pH4.3 and pH 3.0, BLG was substantially rejected by the 300 kDa positivelycharged ultrafiltration membrane because the isoelectric point of BLG(pl 5.1) was greater than either pH value, and BLG carried a substantialnet positive charge. If BLG was the protein of interest, then theprotein mixture would be adjusted to about pH 5.1 to render a net chargeof about zero on the BLG while the other proteins carry a substantialnet negative charge. This would allow BLG to freely pass through a 300kDa negatively charged ultrafiltration membrane while the other proteinswould not.

What is surprising about these results is that by using a chargedultrafiltration membrane, proteins more than ten times smaller than therated pore size of membrane can be fractioned from each other. Thisdiscovery offers the advantage of making dairy protein fractions ofchromatographic purity without the need for sophisticated chromatographyequipment or water or buffers. Furthermore, the water flux of a 300 kDamembrane is more than 60 times greater than the flux of a 10 kDamembrane; the pore size one would expect to be needed to fractionateproteins of size 8.6 to 18.6 kDa. Higher flux corresponds to fasterproduction rates for protein purification and a dramatically lower costof manufacture.

VI: CONCLUSION

All the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   -   U.S. Pat. No. 4,824,568    -   U.S. Pat. No. 4,849,106    -   U.S. Patent Publn. No. 2002/0185440    -   U.S. Patent Publn. No. 2003/0178368 A1

What is claimed is:
 1. A method for fractionating a protein mixture, themethod comprising: (a) adjusting the pH of the protein mixture based onan isoelectric point of a protein of interest in the protein mixture,thereby rendering a net charge of about zero on the protein of interest;(b) adjusting conductivity of the protein mixture such that proteinsother than the protein of interest are rejected by a chargedultrafiltration membrane; and (c) contacting the mixture with a chargedultrafiltration membrane to achieve a first permeate and a firstretentate, wherein the ultrafiltration membrane has a pore size at least10× greater than the molecular mass of at least one of the proteinsother than the protein of interest, and has a pore size of between about86 kDa and about 1,600 kDa, wherein the first permeate comprises anincreased ratio of the protein of interest as compared to the proteinmixture.
 2. The method of claim 1, wherein the protein mixture is a milkprotein or a whey protein mixture.
 3. The method of claim 1, furthercomprising subjecting the first permeate to a second chargedultrafiltration to achieve a second permeate and a second retentate. 4.The method of claim 3, wherein the second retentate is recycled intoanother protein mixture for additional charged ultrafiltration.
 5. Themethod of claim 3, wherein the ultrafiltration achieves a purity ofabout 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%.
 6. The method ofclaim 3, wherein the ultrafiltration achieves a yield of about 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% or 99%.
 7. The method of claim 1, furthercomprising subjecting the first retentate to a second chargedultrafiltration to achieve a second retentate and a second permeate. 8.The method of claim 7, wherein the second permeate is recycled intoanother protein mixture for additional charged ultrafiltration.
 9. Themethod of claim 7, wherein the ultrafiltration achieves a purity ofabout 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%.
 10. The method ofclaim 7, wherein the ultrafiltration achieves a yield of about 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% or 99%.
 11. The method of claim 1, whereinthe ultrafiltration achieves a purity of about 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% or 99%.
 12. The method of claim 1, wherein theultrafiltration achieves a yield of about 60%, 65%, 70%, 75%, 80%, 85%,90%, 95% or 99%.
 13. The method of claim 3, wherein the ultrafiltrationmembrane is positively charged or negatively charged.
 14. The method ofclaim 1, wherein the conductivity is adjusted to 3-10 mS/cm.
 15. Themethod of claim 1, wherein GMP is separated from ALA, IgG, or BLG; orwherein ALA is separated from IgG or BLG; or wherein BLG is separatedfrom IgG.
 16. The method of claim 1, wherein the charged ultrafiltrationis effected by a multistage cross-flow positively chargedultrafiltration membrane.